Primer compositions and multiplex methods for detecting the presence of listeria

Abstract

Aspects of the disclosure relate to primer compositions and multiplex methods for detecting the presence of Listeria. The primer composition includes a first primer sequence operable to amplify a first polymerase chain reaction (PCR) product (e.g., a nucleic acid, a fragment thereof, a complement thereof, or a gene). The first primer sequence is specific for the Listeria genus. The primer composition also includes at least one additional primer sequence operable to amplify at least one additional PCR product. The additional primer sequence(s) are specific for one or more species or subspecies of Listeria. The multiplex method includes contacting a sample with the primer composition to substantially simultaneously detect the presence of the Listeria genus and differentiate between species of Listeria present in the sample.

What is claimed is: 
     
         1 . A primer composition comprising:
 a first primer set operable to amplify a first nucleic acid, a fragment thereof, a complement thereof, or a gene, the first primer set including at least a first primer sequence and a second primer sequence, the at least a first primer sequence and a second primer sequence specific for genus  Listeria;      a second primer set operable to amplify at least a second nucleic acid, a fragment thereof, or a complement thereof, or a gene, the second primer set including at least a third primer sequence and a fourth primer sequence, the at least a third primer sequence and a fourth primer sequence specific for at least one of a first species of  Listeria  or a subspecies thereof; and   at least a third primer set operable to amplify at least a third nucleic acid, a fragment thereof, or a complement thereof, or a gene, the at least a third primer set including at least a fifth primer sequence and a sixth primer sequence, the at least a fifth primer sequence and a sixth primer sequence specific for at least one of a second species of  Listeria  or a subspecies thereof.   
     
     
         2 . The primer composition of  claim 1 , wherein each of the at least a first primer sequence and at least a second primer sequence comprise at least one of a nucleic acid molecule having a sequence comprised in SEQ ID NO: 12 and SEQ ID NO: 13, a nucleic acid molecule with at least 90% sequence homology thereto, or a nucleic acid molecule complementary thereto. 
     
     
         3 . The primer composition of  claim 1 , wherein each of the at least a third primer sequence and at least a fourth primer sequence comprise at least one of a nucleic acid molecule having a sequence comprised in SEQ ID NO: 1 through SEQ ID NO: 11, a nucleic acid molecule with at least 90% sequence homology thereto, or a nucleic acid molecule complementary thereto. 
     
     
         4 . The primer composition of  claim 1 , wherein each of the at least a fifth primer sequence and at least a sixth primer sequence comprise at least one of a nucleic acid molecule having a sequence comprised in SEQ ID NO: 1 through SEQ ID NO: 11, a nucleic acid molecule with at least 90% sequence homology thereto, or a nucleic acid molecule complementary thereto. 
     
     
         5 . The primer composition of  claim 1 , wherein the at least a third primer sequence and a fourth primer sequence comprise at least one of a set of sequences comprised of at least one of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 through SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, or SEQ ID NO: 10 and SEQ ID NO: 11, a set of nucleic acid molecules with at least 90% sequence homology to the set of sequences, a set of nucleic acid molecules complementary to the set of sequences, or a set of nucleic acid molecules complementary to the nucleic acid molecules with at least 90% sequence homology to the set of sequences. 
     
     
         6 . The primer composition of  claim 1 , wherein the at least a third primer sequence and a fourth primer sequence are specific for at least one of  L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri , or  L. grayi.    
     
     
         7 . A method for determining the presence of  Listeria  organism in a sample, the method comprising:
 contacting the sample with a primer composition, the primer composition including:
 a first primer set operable to amplify a first nucleic acid, a fragment thereof, a complement thereof, or a gene; 
 at least one additional primer set operable to amplify at least a second nucleic acid, a fragment thereof, a complement thereof, or a gene; 
   detecting at least one of the first amplified nucleic acid, a fragment thereof, a complement thereof, or a gene, and the at least a second amplified nucleic acid, a fragment thereof, a complement thereof, or a gene, wherein detecting the first amplified nucleic acid, a fragment thereof, a complement thereof, or a gene is indicative of the presence of genus  Listeria  in the sample, and detecting the at least a second amplified nucleic acid, a fragment thereof, a complement thereof, or a gene is indicative of at least one of a species of  Listeria  or a subspecies thereof in the sample.   
     
     
         8 . The method for determining the presence of  Listeria  organism in a sample of  claim 7 , wherein the first primer set comprises at least one of a pair of nucleic acid molecules having the sequences comprised in SEQ ID NO: 12 and SEQ ID NO: 13, a pair of nucleic acid molecules with at least 90% sequence homology thereto, or a pair of nucleic acid molecules complementary thereto. 
     
     
         9 . The method for determining the presence of  Listeria  organism in a sample of  claim 7 , wherein the at least one additional primer set comprises at least one of a pair of nucleic acid molecules having sequences comprised in SEQ ID NO: 1 through SEQ ID NO: 11, a pair of nucleic acid molecules with at least 90% sequence homology thereto, or a pair of nucleic acid molecules complementary thereto 
     
     
         10 . The method for determining the presence of  Listeria  organism in a sample of  claim 7 , wherein the at least one additional primer set is operable to amplify at least a third nucleic acid, a fragment thereof, a complement thereof, or a gene. 
     
     
         11 . The method for determining the presence of  Listeria  organism in a sample of  claim 10 , further comprising detecting the at least a third amplified nucleic acid, fragment thereof, or complement thereof, wherein detecting the at least a third amplified nucleic acid, a fragment thereof, a complement thereof, or a gene is indicative of at least one of an additional species of  Listeria  or an additional subspecies of  Listeria.    
     
     
         12 . The method for determining the presence of  Listeria  organism in a sample of  claim 7 , wherein detecting the at least a second amplified nucleic acid, a fragment thereof, a complement thereof, or a gene is indicative of the presence of at least one of  L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri , or  L. grayi  in the sample. 
     
     
         13 . The method for determining the presence of  Listeria  organism in a sample of  claim 7 , wherein the detecting is performed by a PCR technique. 
     
     
         14 . The method for determining the presence of  Listeria  organism in a sample of  claim 13 , wherein the PCR technique comprises at least one of agarose gel assay or a real-time PCR technique. 
     
     
         15 . The method for determining the presence of  Listeria  organism in a sample of  claim 7 , further comprising subjecting the sample to a series of PCR cycles configured to anneal the at least a first primer set to the at least a first nucleic acid, a fragment thereof, a complement thereof, or a gene and to anneal the at least a second primer set to the at least a second nucleic acid, a fragment thereof, a complement thereof, or a gene. 
     
     
         16 . A primer composition comprising:
 a first primer set operable to amplify a first nucleic acid, a fragment thereof, a complement thereof, or a gene, the first primer set comprising at least one of a first set of sequences comprised of SEQ ID NO: 12 and SEQ ID NO: 13, a set of nucleic acid molecules with at least 90% sequence homology to the first set of sequences, a set of nucleic acid molecules complementary to the first set of sequences, or a set of nucleic acid molecules complementary to the nucleic acid molecules with at least 90% sequence homology to the first set of sequences; and   at least one additional primer set operable to amplify at least a second nucleic acid, a fragment thereof, a complement thereof, or a gene, the at least one additional primer set comprising at least one of a second set of sequences comprised of at least one of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 through SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, or SEQ ID NO: 10 and SEQ ID NO: 11, a set of nucleic acid molecules with at least 90% sequence homology to the second set of sequences, a set of nucleic acid molecules complementary to the second set of sequences, or a set of nucleic acid molecules complementary to the nucleic acid molecules with at least 90% sequence homology to the second set of sequences.   
     
     
         17 . The primer composition of  claim 16 , wherein the first primer set is specific for genus  Listeria.    
     
     
         18 . The primer composition of  claim 16 , wherein the at least one additional primer set is specific for at least one of  L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri , or  L. grayi.    
     
     
         19 . The primer composition of  claim 16 , wherein the at least one additional primer set further comprises:
 a second primer set, the second primer set operable to amplify the at least a second nucleic acid, a fragment thereof, a complement thereof, or a gene, the second primer set comprising at least one of a second set of sequences comprised of at least one of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 through SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, or SEQ ID NO: 10 and SEQ ID NO: 11, a set of nucleic acid molecules with at least 90% sequence homology to the second set of sequences, a set of nucleic acid molecules complementary to the second set of sequences, or a set of nucleic acid molecules complementary to the nucleic acid molecules with at least 90% sequence homology to the second set of sequences; and   a third primer set, the third primer set operable to amplify at least a third nucleic acid, a fragment thereof, a complement thereof, or a gene, the third primer set comprising at least one of a third set of sequences different than the second set of sequences, the third set of sequences comprised of at least one of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 through SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, or SEQ ID NO: 10 and SEQ ID NO: 11, a set of nucleic acid molecules with at least 90% sequence homology to the third set of sequences, a set of nucleic acid molecules complementary to the third set of sequences, or a set of nucleic acid molecules complementary to the nucleic acid molecules with at least 90% sequence homology to the third set of sequences.   
     
     
         20 . The primer composition of  claim 16 , wherein the second primer set is specific for a first species of  Listeria  or subspecies of thereof, and the third primer set is specific for a second species of  Listeria  or subspecies of thereof.